Sheep model for osteoporosis: Sustainability and biomechanical relevance of low turnover osteoporosis induced by hypothalamic–pituitary disconnection

Hypothalamo‐pituitary disconnection (HPD) leads to low bone turnover and osteoporosis in sheep. To determine the sustainability of bone loss and its biomechanical relevance, we studied HPD‐sheep 24 months after surgery (HPD + OVX‐24) in comparison to untreated control (Control), ovariectomized sheep (OVX), and sheep 12 months after HPD (HPD + OVX‐12). We performed histomorphometric, HR‐pQCT, and qBEI analyses, as well as biomechanical testing of all ewes studied. Twenty‐four months after HPD, histomorphometric analyses of the iliac crest showed a significant reduction of BV/TV by 60% in comparison to Control. Cortical thickness of the femora measured by HR‐pQCT did not change between 12 and 24 months after HPD but remained decreased by 30%. These structural changes were caused by a persisting depression of osteoblast and osteoclast cellular activity. Biomechanical testing of the femora showed a significant reduction of bending strength, whereas calcium content and distribution was found to be unchanged. In conclusion, HPD surgery leads to a persisting low turnover status with negative turnover balance in sheep followed by dramatic cortical and trabecular bone loss with consequent biomechanical impairment. © 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 31:1067–1074, 2013     

Low turnover osteoporosis in sheep induced by hypothalamic-pituitary disconnection

The hypothalamus is of critical importance in regulating bone remodeling. This is underscored by the fact that intracerebroventricular-application of leptin in ewe leads to osteopenia. As a large animal model of osteoporosis, this approach has some limitations, such as high technical expenditure and running costs. Therefore we asked if a surgical ablation of the leptin signaling axis would have the same effects and would thereby be a more useful model. We analyzed the bone phenotype of ewe after surgical hypothalamo-pituitary disconnection (HPD + OVX) as compared to control ewe (OVX) after 3 and 12 months. Analyses included histomorphometric characterization, micro-CT and measurement of bone turnover parameters. Already 3 months after HPD we found osteopenic ewe with a significantly decreased bone formation (69%) and osteoclast activity (49%). After a period of 12 months the HPD group additionally developed an (preclinical) osteoporosis with significant reduction (33%) of femoral cortical thickness, as compared to controls (OVX). Taken together, HPD leads after 12 month to osteoporosis with a reduction in both trabecular and cortical bone caused by a low bone turnover situation, with reduced osteoblast and osteoclast activity, as compared to controls (OVX). The HPD-sheep is a suitable large animal model of osteoporosis. Furthermore our results indicate that an intact hypothalamo-pituitary axis is required for activation of bone turnover.     

Effect of low molecular weight heparin on fracture healing in a stabilized rat femur fracture model.

The purpose of this study was to evaluate the effect of low molecular weight heparin (LMWH) on fracture healing in a standard stabilized rat femur fracture model. A closed, mid-diaphyseal transverse fracture was created in the right femur of Long-Evans rats after insertion of a 0.8-mm K-wire into the medullary canal. Animals were randomized to receive either LMWH (70 units/kg dalteparin) or an injection of normal saline daily for 2 weeks. Animals were sacrificed at 2, 3, and 6 weeks. Fracture healing was assessed by radiographs, histology, and mechanical testing. There were no significant differences between the control and LMWH groups in the percentage of animals with radiographic bridging callus at each time point. Histologic appearance of fracture healing was similar between the control and LMWH groups. There were no significant differences in the normalized mechanical properties of the control and LMWH groups at 2 and 3 weeks. At 6 weeks, the percent torque of the LMWH group was significantly greater than the control group ( p = 0.0072), however, there was no significant difference in the stiffness and energy absorption. Dalteparin, at the dosage used in this study, did not impair fracture healing in this standard stabilized rat femur fracture model.     

Comparison of the effects of once‐weekly and once‐daily rhPTH (1–34) injections on promoting fracture healing in rodents

To compare the efficacy of once‐weekly and once‐daily subcutaneous injections of teriparatide (recombinant human parathyroid hormone 1–34) on fracture healing, 50 adult male Sprague–Dawley rats were subjected to a unilateral tibia fracture and received internal fixation with a Kirschner needle. Based on the injection dose and frequency, the rats were randomly divided into five groups (n = 10 each): subcutaneous injections of saline or 10 µg/kg/w, 20 µg/kg/w, 10 µg/kg/d, and 20 µg/kg/d teriparatide. Four weeks later, the rats were euthanatized, and the fractured tibiae were assessed using X‐rays, dual‐energy X‐ray absorptiometry, micro‐computed tomography, the three‐point bending biomechanics test, and histology. Compared to the saline control group, either daily or weekly subcutaneous injections of teriparatide significantly increased bone mass, improved the bone microarchitecture, and promoted fracture healing (p < 0.05). There were no significant differences in bone mineral density (BMD), bone microstructure or bone strength between the 20 µg/kg/w and 10 µg/kg/d groups (p > 0.05). Teriparatide 20 µg weekly injections promoted bone fracture healing to the same extent as teriparatide 10 µg daily injections, which can dramatically decrease the cumulative dosage of teriparatide injections. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1145–1152, 2018.

     

Sheep model for osteoporosis: The effects of peripheral hormone therapy on centrally induced systemic bone loss in an osteoporotic sheep model

Hypothalamic-pituitary disconnection (HPD) leads to low bone turnover followed by bone loss and reduced biomechanical properties in sheep. To investigate the role of peripheral hormones in this centrally induced systemic bone loss model, we planned a hormone replacement experiment. Therefore, estrogen (OHE), thyroxin (OHT) or a combination of both (OHTE) was substituted in ovariectomized HPD sheep, as both hormones are decreased in HPD sheep and are known to have a significant but yet not fully understood impact on bone metabolism. Bone turnover and structural parameters were analyzed in comparison to different control groups – untreated sheep (C), ovariectomized (O) and ovariectomized + HPD sheep (OH). We performed histomorphometric and HR-pQCT analyses nine months after the HPD procedure, as well as biomechanical testing of all ewes studied. In HPD sheep (OH) the low bone turnover led to a significant bone loss. Treatment with thyroxin alone (OHT) mainly increased bone resorption, leading to a further reduction in bone volume. In contrast, the treatment with estrogen alone (OHE) and the combined treatment with estrogen and thyroxin (OHTE) prevented HPD-induced bone loss completely. In conclusion, peripheral hormone substitution was able to prevent HPD-induced low-turnover osteoporosis in sheep. But only the treatment with estrogen alone or in combination with thyroxin was able to completely preserve bone mass and structure. These findings demonstrate the importance of peripheral hormones for a balanced bone remodeling and a physiological bone turnover.     

Combined application of low‐intensity pulsed ultrasound and functional electrical stimulation accelerates bone–tendon junction healing in a rabbit model

The objective of this study was to elucidate the combined use of low‐intensity pulsed ultrasound (LIPUS) and functional electrical stimulation (FES) on patella–patellar tendon (PPT) junction healing using a partial patellectomy model in rabbits. LIPUS was delivered continuously starting day 3 postoperative until week 6. FES was applied on quadriceps muscles to induce tensile force to the repaired PPT junction 5 days per week for 6 weeks since week 7 postoperatively. Forty rabbits with partial patellectomy were randomly divided into four groups: control, LIPUS alone, FES alone, and LIPUS + FES groups. At week 12, the PPT complexes were harvested for histology, radiographs, peripheral quantitative computed tomography, and biomechanical testing. There was better remodeling of newly formed bone and fibrocartilage zone in the three treatment groups compared with the control group. LIPUS and/or FES treatments significantly increased the area and bone mineral content of new bone. The failure load and ultimate strength of PPT complex were also highly improved in the three treatment groups. More new bone formed and higher tensile properties were showed in the LIPUS + FES group compared with the LIPUS or FES alone groups. Early LIPUS treatment and later FES treatment showed the additive effects of accelerating PPT junction healing. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:204–209, 2014.     

Evaluation of bone turnover markers and serum minerals variations for predicting fracture healing versus non-union processes in adult sheep as a model for orthopedic research

Bone turnover markers (BTMs) have been considered as an auxiliary method of following the fracture healing process and for early prediction of impaired bone healing. A better understanding of the potential of BTMs in this application could allow for earlier interventions and improved patient care. The aim of this study with a large animal experimental model was to assess the variation of bone formation markers − namely the total alkaline phosphatase (ALP) and its bone-specific isoform (BALP), serum concentration of intact osteocalcin (OC), N-terminal propeptide type III procollagen (PIIINP) and of bone resorption markers – namely tartrate resistant acid phosphatase (TRAP) and deoxypyridinoline crosslink (DPD) during the first stages of a normal fracture healing process and of a segmental critical size defect (CSD), which progresses to a non-union process. Thirty healthy female sheep (Portuguese Churra-da-Terra-Quente breed), approximately 4-years-old, were enrolled in this study. Jugular venous blood samples were collected pre-operatively and at 1, 2, 3, 4, 6, 8, 10 and 12 post-operative weeks. The animals of the CSD group showed significant lower serum levels of BALP, OC and significant higher serum PIIINP levels at early stages of the fracture healing process, compared with animals that progressed in a normal fracture healing process. Serum BALP, OC and PIIINP levels could be useful as non-invasive auxiliary tools with other complementary methods for predicting the outcome of traumatic bone fractures.     

BMP‐7–induced ectopic bone formation and fracture healing is impaired by systemic NSAID application in C57BL/6‐mice

Nonsteroidal antiinflammatory drugs (NSAIDs) are known to potentially impair the fracture healing process. The aim of the present study was to determine if the impairment of bone healing by systemic NSAID application is, at least in part, due to an interaction of NSAIDs with the bone anabolic BMP‐7 pathway. Therefore, we first analyzed fracture healing in control and diclofenac‐treated mice, where we not only found a significant impairment of fracture healing due to diclofenac treatment as assessed by biomechanical testing and µCT imaging, but also found high coexpression of bone morphogenetic protein‐7 (BMP‐7) and cyclooxygenase‐2 (COX‐2) within the fracture callus of both groups. To experimentally address the possible interaction between BMP‐7 and COX‐2, we then induced ectopic bone formation in control (n = 10) and diclofenac‐treated mice (n = 10) by application of BMP‐7 (recombinant human OP‐1, rhOP‐1) into the hamstring muscles. After 20 days of treatment, each ectopic bone nodule was analyzed by contact‐radiography, µCT, histology, and histomorphometry. Diclofenac application decreased the trabecular number and bone mass in the ectopic bone nodules significantly due to reduced osteoblast number and activity. These data demonstrate that the bone anabolic effect of BMP‐7 and fracture healing is impaired by diclofenac application, and suggest that the potential negative impact of NSAIDs on fracture healing is, at least in part, due to interference with BMP‐7 signaling. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:785–791, 2010     

PTH(1–84) administration reverses abnormal bone‐remodeling dynamics and structure in hypoparathyroidism

Hypoparathyroidism is associated with abnormal structural and dynamic skeletal properties. We hypothesized that parathyroid hormone(1–84) [PTH(1–84)] treatment would restore skeletal properties toward normal in hypoparathyroidism. Sixty‐four subjects with hypoparathyroidism were treated with PTH(1–84) for 2 years. All subjects underwent histomorphometric assessment with percutaneous iliac crest bone biopsies. Biopsies were performed at baseline and at 1 or 2 years. Another group of subjects had a single biopsy at 3 months, having received tetracycline before beginning PTH(1–84) and prior to the biopsy (quadruple‐label protocol). Measurement of biochemical bone turnover markers was performed. Structural changes after PTH(1–84) included reduced trabecular width (144 ± 34 µm to 128 ± 34 µm, p = 0.03) and increases in trabecular number (1.74 ± 0.34/mm to 2.07 ± 0.50/mm, p = 0.02) at 2 years. Cortical porosity increased at 2 years (7.4% ± 3.2% to 9.2% ± 2.4%, p = 0.03). Histomorphometrically measured dynamic parameters, including mineralizing surface, increased significantly at 3 months, peaking at 1 year (0.7% ± 0.6% to 7.1% ± 6.0%, p = 0.001) and persisting at 2 years. Biochemical measurements of bone turnover increased significantly, peaking at 5 to 9 months of therapy and persisting for 24 months. It is concluded that PTH(1–84) treatment of hypoparathyroidism is associated with increases in histomorphometric and biochemical indices of skeletal dynamics. Structural changes are consistent with an increased remodeling rate in both trabecular and cortical compartments with tunneling resorption in the former. These changes suggest that PTH(1–84) improves abnormal skeletal properties in hypoparathyroidism and restores bone metabolism toward normal euparathyroid levels. © 2011 American Society for Bone and Mineral Research     

Evaluation of Hyphecan (1‐4,2‐acetamide‐deoxy‐B‐D‐glucan polymer) on wound healing in a rodent model

Objective: To evaluate the effect of an artificial skin Hyphecan (1‐4,2‐acetamide‐deoxy‐B ‐D ‐glucan polymer) on wound healing in a rodent model. Materials and Method: The prospective study was conducted at a basic science laboratory at a tertiary teaching hospital. Two 4 cm × 4 cm full‐thickness wounds were created on the dorsal surface of 10 Spraque–Dawley rats and covered with Hyphecan and Kaltostat, respectively. Wounds were examined and measured on days 4, 10, 21 and 28, and would continue after day 28 until healed up completely. Punch biopsies (3 mm) were taken on days 4, 10 and 28 for histological examination of the response of healing and repair. Results: Despite the fact that the wound healing rate was similar for both groups on days 4, 10, 21 and 28, the average healing time for the Hyphecan group (29.1 ± 1.7 days) was significantly shorter statistically (P = 0.03) than the Kaltostat group (30.7 ± 2.8 days). Conversely, the marked healing response elicited by Hyphecan on day 4 persisted on days 10 and 28 in contrast to Kaltostat, which had only a mild degree of healing response on days 10 and 28. The study suggests that wounds treated by Hyphecan heal faster than Kaltostat. Conclusion: The findings provide basic scientific evidence supporting the clinical use of Hyphecan in different wounds and might also reduce the cost of wound management as Hyphecan is cheaper than Kaltostat and requires a shorter treatment time.      

The GDF5 mutant BB-1 enhances the bone formation induced by an injectable,poly(l-lactide-co-glycolide) acid (PLGA) fiber-reinforced,brushite-forming cement in a sheep defect model of lumbar osteopenia

Background Context

Targeted delivery of osteoinductive bone morphogenetic proteins (eg, GDF5) in bioresorbable calcium phosphate cement (CPC), potentially suitable for vertebroplasty and kyphoplasty of osteoporotic vertebral fractures, may be required to counteract augmented local bone catabolism and to support complete bone regeneration. The biologically optimized GDF5 mutant BB-1 may represent an attractive drug candidate for this purpose.

Changes in oxidative metabolism of murine spleen following laser and superluminous diode (660–950 nm) irradiation: Effects of cellular composition and radiation parameters

Chemiluminescence (CL) of splenocytes of A/Sn mice was recorded after irradiation of the cells with various individual laser an superluminous diode probes at wavelengths from 660 to 950 nm (pulse repetition rates varying from 4 to 5,000 Hz) and at various doses. Laser radiation was found to increase or suppress the spontaneous CL of splenocytes suspension, the amplitude and the sign of the effect depending on the cellular composition of the samples. Direct correlations between the effect of laser radiation (per cent in changes of CL when irradiated at 820 nm, 1.1.10³ J/m², 292 Hz) and per cent of plasmacytes (r = 0.743, P<0.001), neutrophils (r = 0.650, P<0.001) as well as myelocytes and metamyelocytes (r = 0.505, P<0.01) were established. The correlation with per cent of lymphocytes (r = ?0.590, P<0.001) was found to be a reverse one. Dependence of the irradiation effects on dose, pulse repetition rate, and wavelength are presented. © 1993 Wiley-Liss, Inc.